Undifferentiated Embryonic Stem Research Articles

Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

SARS-CoV-2 Mutations Lead to a Decrease in the Number of Tissue-Specific MicroRNA-Binding Regions in the Lung.

RNA interference in vertebrates acts as an antiviral mechanism only in undifferentiated embryonic stem cells and is mediated by microRNAs. In somatic cells, host microRNAs also bind to the genomes of RNA viruses, regulating their translation and replication. It has been shown that viral (+)RNA can evolve under the influence of host cell miRNAs. In more than two years of the pandemic, the SARS-CoV-2 virus has mutated significantly. It is quite possible that some mutations could be retained in the virus genome under the influence of miRNAs produced by alveolar cells. We demonstrated that microRNAs in human lung tissue exert evolutionary pressure on the SARS-CoV-2 genome. Moreover, a significant number of sites of host microRNA binding with the virus genome are located in the NSP3-NSP5 region responsible for autoproteolysis of viral polypeptides.

Transcriptome profiles associated with human periodontal ligament differentiation

ObjectivesTissue differentiation is regulated by transcription factors. This study aimed to identify candidate transcription factors that induce periodontal ligament (PDL) cell differentiation in human pluripotent stem cells (hPSCs). MethodsHuman PDL tissues were scraped from the root surfaces of extracted teeth for orthodontic treatment and cultured using the explant culture method. We used RNA-seq to generate gene expression profiles of third-passage PDL cells and compared them with those of undifferentiated human induced pluripotent stem cells (hiPSCs) and human embryonic stem cell (hESC)-derived neural crest (NC) cells (publicly available data). ResultsPrimary cultured PDL cells exhibited a spindle-shaped fibroblast-like appearance and the gene expression of several PDL cell-specific markers. The gene expression profiles of PDL cells were relatively similar to those of hESC-derived NC cells but not those of undifferentiated hiPSCs. Thirty-seven transcription factors were identified as upregulated genes in PDL cells. Pathway analysis showed that differentially expressed genes were enriched in several functional groups and pathways, including the SMAD 2/3 nuclear pathway. ConclusionsWe identified 37 upregulated transcription genes in primary cultured PDL cells compared with hESC-derived NC cells. Regulating these genes and the SMAD signaling pathway may be promising ways to induce PDL cells from hPSC-derived NC cells.

Directed Differentiation of Human Pluripotent Stem Cells to Cytotrophoblast and Syncytiotrophoblast.

Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs. CTBs are the trophoblast stem cells that can differentiate into specialized placental cell types such as STBs. The multinucleated STB plays vital role in the exchange of nutrients and gases across the placenta and secretes several hormones during pregnancy, such as human chorionic gonadotropin β (hCGβ). Here we describe two methods of seeding the hPSCs: chemical (clumps method) and enzymatic methods (single cells) to differentiate them to CTB and STB, activating BMP (B) signaling and inhibiting ACTIVIN/NODAL and FGF signaling pathways (2i), thus naming our protocol as "B2i" (Sudheer et al., Stem Cells Dev 21:2987-3000, 2012). This protocol forms the perfect model system for understanding in vitro placentation, modeling diseases arising from abnormal placentation that cause complications such as miscarriage, preeclampsia or intrauterine growth restriction (IUGR), and drug discovery for placental disorders.

Changes in the Expression of Pyruvate Dehydrogenase Kinase Isoenzymes During Early Differentiation of Mouse Embryonic Stem Cells

The embryonic stem cells (ESCs) are pluripotent, self-renewing cells that able to differentiate into any of the germ layers involved in embryogenesis. However, the molecular mechanisms that control ESC pluripotency and differentiation remain poorly understood. The family of Pyruvate dehydrogenase kinase (PDK1-4), inactivates the mitochondrial pyruvate dehydrogenase complex via phosphorylation, plays a crucial role in the control of glucose homeostasis. In the current study, gene expression levels of PDK isoenzymes were analyzed on undifferentiated mouse embryonic stem cells (mESCs) and compared to mESCs induced to differentiate by removal of leukemia inhibitory factor (LIF) for 1, 3 and 5 days. Besides, we performed gene expression analysis of several genes related to pluripotency and differentiation. In addition, we also determined glucose uptake rates by a colorimetric assay kit in early differentiated and undifferentiated mESCs. Differently expression level of PDK isoenzymes in pluripotent and LIF-depleted mESCs suggest that they may have roles in differentiation and pluripotency of ESCs. Furthermore, this study lays the foundation for detailed investigation of molecular mechanisms underlying the roles of PDKs in the pluripotency and transition to differentiated state of ESCs.

Maternal Undernutrition Induces Cell Signalling and Metabolic Dysfunction in Undifferentiated Mouse Embryonic Stem Cells

Peri-conceptional environment can induce permanent changes in embryo phenotype which alter development and associate with later disease susceptibility. Thus, mouse maternal low protein diet (LPD) fed exclusively during preimplantation is sufficient to lead to cardiovascular, metabolic and neurological dysfunction in adult offspring. Embryonic stem cell (ESC) lines were generated from LPD and control NPD C57BL/6 blastocysts and characterised by transcriptomics, metabolomics, bioinformatics and molecular/cellular studies to assess early potential mechanisms in dietary environmental programming. Previously, we showed these lines retain cellular and epigenetic characteristics of LPD and NPD embryos after several passages. Here, three main changes were identified in LPD ESC lines. First, their derivation capacity was reduced but pluripotency marker expression was similar to controls. Second, LPD lines had impaired Mitogen-activated protein kinase (MAPK) pathway with altered gene expression of several regulators (e.g., Maff, Rassf1, JunD), reduced ERK1/2 signalling capacity and poorer cell survival characteristics which may contribute to reduced derivation. Third, LPD lines had impaired glucose metabolism comprising reduced upstream enzyme expression (e.g., Gpi, Mpi) and accumulation of metabolites (e.g., glucose-6-P, fructose-6-P) above the phosphofructokinase (PFK) gateway with PFK enzyme activity reduced. ESC lines may therefore permit investigation of peri-conceptional programming mechanisms with reduced need for animal experimentation.Graphical

StemPanTox: A fast and wide-target drug assessment system for tailor-made safety evaluations using personalized iPS cells

SummaryAn alternative model that reliably predicts human-specific toxicity is necessary because the translatability of effects on animal models for human disease is limited to context. Previously, we developed a method that accurately predicts developmental toxicity based on the gene networks of undifferentiated human embryonic stem (ES) cells. Here, we advanced this method to predict adult toxicities of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, two types of nephrotoxins, and non-genotoxic carcinogens) and achieved high predictability (AUC = 0.90–1.00) in all categories. Moreover, we screened for an induced pluripotent stem (iPS) cell line to predict the toxicities based on the gene networks of iPS cells using transfer learning of the gene networks of ES cells, and predicted toxicities in four categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) with high performance (AUC = 0.82–0.99). This method holds promise for tailor-made safety evaluations using personalized iPS cells.

POU5F1 Protein and Gene Expression Analysis in Neonate and Adult Mouse Testicular Germ Cells by Immunohistochemistry and Immunocytochemistry.

POU5F1 (POU class 5 homeobox 1) is a transcription factor that is critically involved in the self-renewal of undifferentiated embryonic stem cells. In this present study, we have developed our study to analyze the expression of the POU5F1 in the neonatal and adult mice testis section and isolated spermatogonial stem cells (SSCs). We also examine POU5F1 protein localization by three various kinds of antibodies. In this experimental research, to enhance our understanding of the POU5F1 expression levels, protein localization, and function in testicular germ cell, we used immunohistochemistry, immunocytochemistry, and Fluidigm real-time polymerase chain reaction (RT-PCR) analysis in the mouse testis section and neonatal and adult SSCs, and also we used protein-protein network analysis and gene enrichment analysis for genes involved in testicular development. Counting POU5F1-positive cells represented significantly higher expression (p < 0.05) of POU5F1 in the adult testis in comparison to the neonate. Finally, Fluidigm RT-PCR showed a significant expression (p < 0.05) level of germ cells gene POU5F1 in neonate SSCs (1-2 week) than 16-24 week SSCs. The illustrated results identify POU5F1 as a necessary transcription factor of testicular germ cells and can be supportive for the investigation of the development and differentiation of SSCs.

Visualization and quantitative analyses for mouse embryonic stem cell tracking by manipulating hierarchical data structures using time-lapse confocal microscopy images.

We present a cell tracking method for time-lapse confocal microscopy (3D) images that uses dynamic hierarchical data structures to assist cell and colony segmentation and tracking. During the segmentation, the cell and colony numbers and their geometric data are recorded for each 3D image set. In tracking, the colony correspondences between neighboring frames of time-lapse 3D images are first computed using the recorded colony centers. Then, cell correspondences in the correspondent colonies are computed using the recorded cell centers. The examples show the proposed cell tracking method can achieve high tracking accuracy for time-lapse 3D images of undifferentiated but self-renewing mouse embryonic stem (mES) cells where the number and mobility of ES cells in a cell colony may change suddenly by a colony merging or splitting, and cell proliferation or death. The geometric data in the hierarchical data structures also help the visualization and quantitation of the cell shapes and mobility.

In conversation with Christine Watson.

Christine J. Watson is Professor of Cell and Cancer Biology at the University of Cambridge. Christine obtained her Bachelor's (honors) degree in Biochemistry at the University of Glasgow in 1975 and, after a soujourn in Glauco Tocchini-Valentini's lab at the Institute of Cell Biology, Consiglio Nazionale delle Ricerche in Rome, she undertook a PhD in Molecular Genetics at Imperial College London. During her PhD, she looked at differences in gene expression between differentiated and undifferentiated embryonal carcinoma stem cells, inspiring an early interest in gene expression and cell fate determination. Between 1986 and 1992, Christine undertook three postdoctoral research positions that took her from London back to Scotland, where she was first introduced to mammary gland biology through her work with John Clark at the Roslin Institute in Edinburgh. During her time in the Clark lab, Christine identified a factor - later shown to be STAT5 - that binds to the promoter of the milk protein gene β-lactoglobulin. This prompted further work identifying the key role played by the STAT family of transcription factors in mammary gland development. Shortly afterwards, Christine became a group leader at the Roslin Institute and later relocated to the University of Edinburgh to collaborate with Andrew Wyllie. This led to her recruitment to the University of Cambridge in 1998, where she has remained to date. Over the last two decades, the Watson lab has focused on elucidating the mechanisms underlying lineage commitment of mammary stem and progenitor cells and the regulation of cell death in involuting mammary gland. In this interview, Christine discusses her research highlights and provides a glimpse into her personal interests, as she moves towards retirement.

Characterizing the Low-Dose Effects of Methylmercury on the Early Stages of Embryo Development Using Cultured Human Embryonic Stem Cells.

Background:Global concerns of methylmercury (MeHg) exposure have been raised, especially on its effects on pregnant women. Recent epidemiological studies have revealed associations between maternal blood/hair MeHg concentrations, adverse pregnancy outcomes, and developmental deficits. However, the underlying mechanisms remain unclear.Objectives:In this study, we characterized the effects of MeHg exposure on undifferentiated human embryonic stem cells (hESCs) and extrapolated the effects to human embryonic development.Methods:hESCs were exposed to 0, 1, 5, 10, 50, 100 or MeHg for 24 h or 6 d. Cell adherence and colony formation and expansion were examined under the microscope. Cell attachment, viability/proliferation, apoptosis, stress response, cell cycle, autophagy, and expression of cell lineage marker genes and proteins were measured at the end of exposures.Results:Our results indicated that exposure to nanomolar concentrations of MeHg was associated with a) higher levels of reactive oxygen species (ROS) and hemeoxygenase-1 (HO-1), suggesting increased stress and adaptive responses; b) lower cellular adhesion, viability/proliferation, and colony formation and expansion; c) higher levels of apoptosis, reflected by higher cleaved caspase-3 expression and Annexin V binding; d) higher levels of cytoskeleton protein expression; e) higher rates of G1/S phase cell cycle arrest; and f) autophagy inhibition, as shown by a lower LC3BII/LC3BI ratio and accumulation of SQSTM1 (p62). These outcomes were accompanied by higher expressions of self-renewal genes or proteins or both, including OCT4, SOX2, NANOG, and cytokine receptor IL6ST, as well as pluripotency and the cell fate regulator cyclin D1.Discussion:These results revealed that under the selection pressure of exposure to low doses of MeHg, some hESCs underwent apoptosis, whereas others adapted and survived with enhanced self-renewal gene expression and specific morphological phenotypes. Findings from the present study provide in vitro evidence that low doses of MeHg adversely affect hESCs when exposed during a period of time that models embryonic pre-, during, and early postimplantation stages. https://doi.org/10.1289/EHP7349

Homozygous mutation in MCM7 causes autosomal recessive primary microcephaly and intellectual disability

BackgroundMinichromosomal maintenance (MCM) complex components 2, 4, 5 and 6 have been linked to human disease with phenotypes including microcephaly and intellectual disability. The MCM complex has DNA helicase activity...

Signal Transduction of Unique RAS Family Member towards Cell Survival

Small GTPases of RAS act as central regulators of intracellular signal transduction and translate external stimuli to the various cellular responses. Embryonic stem cell expressed RAS (ERAS) is a member of the RAS family that is specifically expressed in undifferentiated mouse embryonic stem cells, hepatic stellate cells and diverse human tumors, such as gastric, breast, brain, pancreatic, and colorectal tumors. Although ERAS belongs to GTPase family, it is an inefficient enzyme to hydrolyze GTP to GDP. Therefore, it remains mainly in its GTP-bound active form and contributes to sustained signal transduction. In comparison with classical members (HRAS, NRAS and KRAS4B), ERAS is known as a unique member, due to its temporal expression, remarkable amino acid sequence deviations and functional differences. Notably, ERAS has been recently proposed as a potential marker for drug resistance in several human tumors. In this minireview, I compare in great detail the biochemical properties of ERAS with conventional members of the RAS family, and discuss the main ERAS function in the control of the PI3K-AKT-mTORC survival pathway. Targeting this pathway may sensitize ERAS expressing cell populations to chemotherapy.

The role of Elabela in kidney disease.

Elabela, also known as Toddler or Apela, is a recently discovered hormonal peptide containing 32 amino acids. Elabela is a ligand of the apelin receptor (APJ). APJ is a G protein-coupled receptor widely expressed throughout body, and together with its cognate ligand, apelin, it plays an important role in various physiological processes including cardiovascular functions, angiogenesis and fluid homeostasis. Elabela also participates in embryonic development and pathophysiological processes in adulthood. Elabela is highly expressed in undifferentiated embryonic stem cells and regulates endoderm differentiation and cardiovascular system development. During differentiation, Elabela is highly expressed in pluripotent stem cells and in adult renal collecting ducts and loops, where it functions to maintain water and sodium homeostasis. Other studies have also shown that Elabela plays a crucial role in the pathogenesis of kidney diseases. This review addresses the role of Elabela in kidney diseases including renal ischemia/reperfusion injury, hypertensive nephropathy, diabetic nephropathy, and cardiorenal syndrome.

LIF maintains mouse embryonic stem cells pluripotency by modulating TET1 and JMJD2 activity in a JAK2-dependent manner.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse embryonic stem cells (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance.

Carbohydrate 3′-sialyllactose as a novel target for theranostics in pancreatic ductal adenocarcinoma

We previously demonstrated that the carbohydrate 3′-sialyllactose is overexpressed in cancer stem-like cells such as metastatic pancreatic and poorly differentiated gastric cancer cells, and undifferentiated human embryonic stem cells. In this study, we investigated the possibility of 3′-sialyllactose as a target for theranostics in cancers using a recombinant mouse monoclonal antibody r3B1E2 that binds to 3′-sialyllactose. Immunohistochemistry analysis confirmed an elevated expression of 3′-sialyllactose in tumors of pancreas, stomach, and testis, while no expression of 3′-sialyllactose was observed in corresponding normal controls. In addition, a stage-independent expression of 3′-sialyllactose was observed, especially in pancreatic ductal adenocarcinoma (PDAC). The level of serum 3′-sialyllactose in PDAC subjects was significantly higher than that in healthy controls, providing excellent AUC of 0.88. We next explored the therapeutic potential of r3B1E2 for PDAC in vitro. Treatment of r3B1E2 with 3′-sialyllactose-bearing human PDAC cells exhibited a complement-dependent cytotoxicity, whereas no significant activity of r3B1E2 against 3′-sialyllactose-negative cells was observed. Collectively, these findings raise the possibility of 3′-sialyllactose as a novel target for theranostics in PDAC.

The H-Y Antigen in Embryonic Stem Cells Causes Rejection in Syngeneic Female Recipients

Pluripotent stem cells are promising candidates for cell-based regenerative therapies. To avoid rejection of transplanted cells, several approaches are being pursued to reduce immunogenicity of the cells or modulate the recipient's immune response. These include gene editing to reduce the antigenicity of cell products, immunosuppression of the host, or using major histocompatibility complex-matched cells from cell banks. In this context, we have investigated the antigenicity of H-Y antigens, a class of minor histocompatibility antigens encoded by the Y chromosome, to assess whether the gender of the donor affects the cell's antigenicity. In a murine transplant model, we show that the H-Y antigen in undifferentiated embryonic stem cells (ESCs), as well as ESC-derived endothelial cells, provokes T- and B cell responses in female recipients.

Genetic switches designed for eukaryotic cells and controlled by serine integrases

Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.

Determination of Glutamate and GABA Released by Mouse Embryonic Stem Cells Using HILIC-ESI-MS/MS

Background:In recent years, more and more researches have shown that neurotransmitters can also be synthesized and released by peripheral non-neural cells. However, specificity and high sensitivity detection means were required for confirming ESCs autocrine glutamate and γ - aminobutyric acid (GABA). Glutamate and GABA are water-soluble and polar compounds which cannot be retained on a reversed phase C18 column, and their contents are often at a trace level. On the other hand, the biological matrix such as cell culture fluid contains a large number of amino acids, vitamins, carbohydrates, inorganic ions and other substances. Therefore, the main problem is the selection of the chromatographic column to avoid matrix interference.Objective:To establish a rapid and reliable method for the simultaneous determination of glutamate and GABA released from embryonic stem cells based on analytical chemistry.Methods:Glutamate and GABA released from mouse embryonic stem cells were determined on the basis of hydrophilic interaction chromatography coupled with electrospray ionization tandem Mass Spectrometry (HILIC- ESI- MS/MS), using isotope internal standards and substitution matrix method.Results:Undifferentiated embryonic stem cells autocrine glutamate and GABA and will reach releasing- reuptacking dynamic equilibriums at different time points. In contrast, neither glutamate nor GABA releasing could be detected from the MEFs, indicating the specificity release of the mESCs in the applied analytic method.Conclusion:A novel, simple, sensitive, selective and quantitative method was developed for determination of the glutamate and GABA from mouse embryonic stem cells.

Isolation and Characterization of Mesenchymal Stem Cells from Human Gingiva

A new nonimmortalized cell line (MSC-GING) was isolated from the gingiva of a 35-year-old healthy donor and characterized. The analysis of different properties was carried out at the 6th, 7th, 13th, 18th, 20th, and 23rd passages. During long-term cultivation, the proportion of aging cells according to the activity of β-galactosidase increased. The cloning efficiency of MSC-GING cells was significantly reduced, and the number of aging β-galactosidase-positive cells was gradually increased during cell prolonged cultivation. Growth curves showed active cell proliferation at the sixth passage. It was significantly declined by the 18th and 20th passages. Karyotypic analysis at the 7th and 18th passages showed the presence of a diploid number of chromosomes (46) in metaphase plates. At passage 7, however, the proportion of cells with a normal diploid karyotype 46, XX, was 50 ± 5%. Other cells had a clonal rearrangement inv (10) (q21.1q25.1), an inversion of the long arm of chromosome 10. Their number significantly decreased during cultivation up to passage 18, while the proportion of cells with a normal diploid karyotype increased. The cell number with surface antigens common for human mesenchymal stem cells (CD44, CD73, CD90, CD105, HLA-ABC) and vimentin was high at passages 6 and 20, while CD34, CD45, and HLA-DR positive cells were very rare. It is noteworthy that no cells carrying markers of undifferentiated embryonic stem cells (Oct-4, SSEA-4, and SOX2) are observed at passages 6 and 20. It was shown that MSC-GING cells were able to differentiate in osteogenic and chondrogenic directions. The ability to differentiate in the adipogenic direction was manifested only at the level of glut4 gene expression. Induction of neuronal differentiation increased the expression the nse gene (neurospecific elonase). In general, the results confirmed the MSC status for the obtained line, but revealed significant karyotypic instability in the early passages, which decreased during the replicative aging.